Lysozyme plays a central role in innate immunity by inhibiting extracellular bacterial viability, particularly on mucosal surfaces such as those of the lung. However, many gram-positive pathogens, including Bacillus anthracis, are resistant in vitro to vertebrate c-type lysozyme, a feature likely relevant for pathogenesis. In Staphylococcus aureus, O-acetylation of the cell wall peptidoglycan has been identified as the modification that is responsible for lysozyme resistance. The presence of a similar modification in B. anthracis is unknown. We hypothesize that the lysozyme resistance of B. anthracis is due to O-acetylation and we will test this hypothesis using established methodology to assay the O-acetylation of B. anthracis peptidoglycan and to characterize the growth phase dependence of this modification. We will identify and characterize the activity of the B. anthracis O-acetyl transferase and determine its membrane topology and subcellular localization. We will assess whether O-acetylation is important in protecting B. anthracis cells from the action of antimicrobial factors including lysozyme secreted by cultured mammalian cells and in their survival during host infection. Unlike the c-type lysozyme expressed by all animals, CH-type lysozyme found in some bacteria and fungi can hydrolyze O-acetylated cell wall. Thus, recombinant CH-type lysozyme may serve as a useful therapeutic agent to supplement host defenses in reducing B. anthracis survival. We have identified genes encoding putative CH-type lysozymes in the genomes of the nematode C. elegans and its close relative C. briggsae, but not in any other sequenced metazoan genome. We hypothesize that these proteins will be capable of hydrolyzing O- acetylated cell walls. We will test this hypothesis by cloning and purifying these enzymes and assessing their ability to digest B. anthracis cell wall and to lyse B. anthracis cells. We will further characterize these enzymes to evaluate their predicted active site residues and substrate specificity. Unlike their bacterial and fungal homologs, these enzymes have evolved to work within the physiological context of a multi-cellular eukaryotic organism, making them excellent candidates as therapeutic agents to overcome B. anthracis lysozyme resistance. PUBLIC HEALTH RELEVANCE We will study the process by which bacteria construct their cell wall, the structure that determines and maintains their shape. Although this process is the target of many presently used antibiotics including penicillin, bacteria are quickly becoming resistant to these drugs, so we expect that new understanding of this process will lead to the development of new antibiotics.